APOBEC3A and APOBEC3B Activities Render Cancer Cells Susceptible to ATR Inhibition Remi Buisson1, Michael S

Published OnlineFirst July 11, 2017; DOI: 10.1158/0008-5472.CAN-16-3389 Cancer Molecular and Cellular Pathobiology Research

APOBEC3A and APOBEC3B Activities Render Cancer Cells Susceptible to ATR Inhibition Remi Buisson1, Michael S. Lawrence1,2, Cyril H. Benes1, and Lee Zou1,3

Abstract

The apolipoprotein B mRNA editing enzyme catalytic poly- ATR-mediated negative feedback loop during replication. The peptide-like APOBEC3A and APOBEC3B have emerged as key surge of abasic sites upon ATR inhibition associated with increas- mutation drivers in cancer. Here, we show that APOBEC3A and ed accumulation of single-stranded DNA, a substrate of APO- APOBEC3B activities impose a unique type of replication stress BEC3A, triggering an APOBEC3A-driven feed-forward loop that by inducing abasic sites at replication forks. In contrast to cells ultimately drove cells into replication catastrophe. In a panel of under other types of replication stress, APOBEC3A-expressing cells cancer cell lines, ATRi selectively induced replication catastrophe in were selectively sensitive to ATR inhibitors (ATRi), but not to a those harboring high APOBEC3A and/or APOBEC3B activities, variety of DNA replication inhibitors and DNA-damaging drugs. showing that APOBEC3A and APOBEC3B activities conferred In proliferating cells, APOBEC3A modestly elicited ATR but susceptibility to ATRi. Our results deﬁne an APOBEC-driven repli- not ATM. ATR inhibition in APOBEC3A-expressing cells resulted cation stress in cancer cells that may offer an opportunity for in a surge of abasic sites at replication forks, revealing an ATR-targeted therapy. Cancer Res; 77(17); 1–12. 2017 AACR.

Introduction APOBEC3A and APOBEC3B (A3A and A3B) are members of the APOBEC family of cytosine deaminases, which convert C to Genomic instability is one of the hallmarks of cancers (1). U in single-stranded DNA (ssDNA; refs. 16–18). Although While genomic instability fuels tumorigenesis, it also offers a APOBEC proteins are important for the cellular defense vulnerability of cancer cells that can be exploited therapeutically. against foreign DNA/RNA during viral infection (19), they DNA replication stress is a major source of genomic instability are not normally expressed in unstressed proliferating cells. (2, 3). Activation of a number of oncogenes, such as MYC, RAS, When A3A and A3B are abnormally expressed in cancer cells, and cyclin E, induces replication stress (4–6). Inactivation of they become potent mutators of the genome. Expression of certain tumor suppressors involved in DNA repair, such as BRCA1 APOBEC3B is prevalent in several cancer types (17, 20–23). In- and BRCA2, also elevates replication stress in cancer cells (7–11). depth analysis of mutation signatures in cancers has implicated Recent cancer genomics studies by others and us revealed that both A3A and A3B in APOBEC-mediated mutagenesis (24). An cancer-associated mutations in the genes encoding mismatch A3AB fusion gene resulting from a deletion in the APOBEC3A- DNA repair proteins and DNA polymerase epsilon (POLE), APOBEC3B locus encodes A3A and associates with increased as well as cancer-associated expression of APOBEC3A and risk for breast and ovarian cancers, and with the APOBEC APOBEC3B, are key drivers of mutation in cancers (12, 13). mutation signature in tumors (25–29). Furthermore, A3A is Notably, the mutations inflicted by these "mutators" display a upregulated in a subset of leukemia (30). When expressed at clear association with either leading or lagging strand of DNA high levels, both A3A and A3B induce DNA double-stranded replication forks, suggesting that they act in a replication-coupled breaks (DSB), and A3A also triggers cell-cycle arrest (17, 18, manner (12, 14, 15). These new findings raise an important 31). Together, these findings show that A3A and A3B are question as to whether these mutators in cancer cells induce important drivers of mutation and genomic instability in a replication stress and, if so, whether the stress induced by them large subset of cancers, raising the question of how cancer cells can be targeted in cancer therapy. cope with these mutators during proliferation, and whether these mutators offer an opportunity for targeted therapy. Here, we show that A3A and A3B impose a unique type of replication stress by cytosine deamination at DNA replication 1Massachusetts General Hospital Cancer Center, Harvard Medical School, Bos- forks. During DNA replication, A3A induces abasic (AP) sites in an 2 ton, Massachusetts. Broad Institute of Harvard and MIT, Cambridge, Massa- UNG2-dependent manner, leading to modest ATR activation. 3 chusetts. Department of Pathology, Massachusetts General Hospital, Harvard Inhibition of ATR in A3A-expressing cells results in a surge of AP Medical School, Boston, Massachusetts. sites at replication forks, revealing a previously unknown ATR- Note: Supplementary data for this article are available at Cancer Research mediated feedback loop that counters A3A. The accumulation of Online (http://cancerres.aacrjournals.org/). AP sites at replication forks upon ATR inhibition increases stalling Corresponding Author: Lee Zou, Massachusetts General Hospital Cancer Cen- of DNA polymerases and exposure of ssDNA, a substrate of A3A. ter, Building 149-7th Floor, 13th Street, Charlestown, MA 02129. Phone: 617-724- In the absence of ATR activity, ssDNA triggers an A3A-driven feed- 9534; Fax: 617-726-7808; E-mail: [email protected] forward loop propelling a further buildup of AP sites and ssDNA doi: 10.1158/0008-5472.CAN-16-3389 at replication forks, which ultimately drives cells into replication 2017 American Association for Cancer Research. catastrophe. Interestingly, the replication stress induced by A3A

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renders cells sensitive to ATR inhibitors (ATRi), but not to a variety Antibodies of replication inhibitors and genotoxic drugs, highlighting the The antibodies used in this study were: gH2AX mAb (Cell unique nature of A3A-induced replication stress and the distinc- Signaling Technology and EMD Millipore), APOBEC3B polyclon- tive role of ATR against this stress. In a panel of cancer cell lines, al antibody (Santa Cruz Biotechnology), Flag-M2 mAb (Sigma- ATRi rapidly induces replication catastrophe in those harboring Aldrich), CDC7 mAb (Abcam), PCNA mAb (Santa Cruz Biotech- high A3A and/or A3B activities, suggesting that the replication nology), Chk1 mAb (Santa Cruz Biotechnology), BrdUrd mAb stress imposed by A3A and A3B may offer a promising opportu- (BD Biosciences), PCNA polyclonal antibody (Abcam), Chk2 nity for ATR-targeted therapy in a variety of cancers. pT68 polyclonal antibody (Cell Signaling Technology), Chk1 pS317 polyclonal antibody (Cell Signaling Technology), GAPDH Materials and Methods polyclonal antibody (EMD Millipore), UNG polyclonal antibody (Novus), RPA32 mAb (Thermo Fisher Scientific), RPA70 poly- Cell lines clonal antibody (Bethyl), APE1 mAb (Santa Cruz Biotechnology), All cell lines were obtained from the Center for Molecular biotin polyclonal antibody (Abcam), and ATR polyclonal anti- Therapeutics (CMT) at the MGH Cancer Center (Boston, MA) body (Bethyl). from 2015 to 2016. The CMT has obtained all cell lines described here from commercial repositories (ATTC, DSMZ, ECACC, or JHSF/JCRB). Upon receipt at CMT, the cell lines Results were expanded and frozen stocks were created. Stocks were APOBEC3A and APOBEC3B activate ATR but not ATM in further authenticated as follows: To identify cross-contami- proliferating cancer cells nated or synonymous lines, a panel of SNPs was profiled for Recentstudiesbyothersandusrevealedthatthemutation each cell line (Sequenom) and a pair-wise comparison score signatures of A3A and A3B are associated with the lagging was calculated. In addition, we performed short tandem strand of DNA replication forks, suggesting that A3A and A3B repeat (STR) analysis (AmpFLSTR Identifiler, Applied Biosys- act on ssDNA during DNA replication (12, 14, 15). To tems) and matched this to an existing STR profile generated by investigate whether A3A and A3B induce replication stress, the providing repository. From authenticated frozen stocks, we analyzed the expression data of A3A and A3B in a large cells were not continuously kept in culture for more than 3 panel of cancer cell lines (Supplementary Fig. S1A). Consistent months. For the experiments described in this article, cell lines with previous studies, A3B mRNA was detected in a large were not continuously kept in culture for more than 3 fraction of cancer cell lines (17, 21). A3A mRNA was also months. All cell lines used in this study were tested for detected in a subset of the cell lines. We selected a group of mycoplasma. cell lines that express A3A and A3B mRNAs at various levels according to the microarray data and then determined the Cell culture relative levels of A3A and A3B mRNAs using qRT-PCR (Sup- U2OS-derived and SKOV3-derived cell lines expressing plementary Fig. S1B). Furthermore, we tested these cell lines in vitro APOBEC3A were generated by infecting U2OS cells with lentivirus for A3A and A3B activities using a previously described – expressing APOBEC3A under a doxycycline-inducible promoter assay (Supplementary Fig. S1C S1E; ref. 17). We measured the (pInducer20) and selected with G418 (400 mg/mL). U2OS deriv- overall A3A-A3B activity because the enzymatic activities of in vitro ative cells were maintained in DMEM supplemented with 10 % A3A and A3B are indistinguishable in this assay. A FBS and 1 % penicillin/streptomycin. For APOBEC3A expression, range of A3A-A3B activities were detected in this group of cell cells were incubated with doxycycline (200 ng/mL) 20 hours lines (Fig. 1A). Notably, although cell lines with high overall before additional treatment. For bromodeoxyuridine (BrdUrd) A3A-A3B activity tend to express high levels of A3A and A3B labeling, cells were incubated with 10 mmol/L BrdUrd for 48 mRNAs, neither A3A nor A3B mRNA alone correlated with hours. OVCAR5, SKOV3, NCI-H2347, and HCC78 were main- overall A3A-A3B activity precisely (Fig. 1A; Supplementary Fig. tained in RPMI1640 GlutaMAX-I supplemented with 10% FBS, S1B), suggesting that both enzymes may contribute to the 1% penicillin/streptomycin, 1% glucose, and 1% sodium pyru- activity in cancer cells. vate. TOV21G, OV17R, BICR6, BHY, HSC4, and BICR31 were Next, we further characterized the three cell lines with the maintained in DMEM/F12 GlutaMAX-I supplemented with 10% highest A3A-A3B activity and the three cell lines with the lowest FBS and 1% penicillin/streptomycin. SKBR3 was maintained in A3A-A3B activity in this panel (Fig. 1A). All these cell lines McCoy's 5A supplemented with 10% FBS and 1% penicillin/ displayed baseline levels of phosphorylated Chk1 (p-Chk1; Fig. – streptomycin. The cell lines above were purchased by the CMT 1B and C), suggesting that the ATR Chk1 pathway is modestly from either ATCC or Sigma-Aldrich. elicited by intrinsic replication stress during proliferation. To determine whether A3A and A3B induce replication stress and contribute to the ATR response, we used multiple siRNAs to knock Inhibitors down both A3A and A3B (Supplementary Fig. S2A–S2C). In The kinase inhibitors used in this study were ATRi (10 mmol/L TOV21G, one of the cell lines harboring high A3A-A3B activity, VE-821, Selleckchem), ATRi#2 (1 mmol/L AZ-20, custom made), A3A-A3B siRNAs eliminated A3A-A3B activity while only slightly Roscovitine (12 mmol/L, Selleckchem), DNA-PKi (2 mmol/L reduced S-phase cells (Supplementary Fig. S2B and S2D). The NU7441, Selleckchem), ATMi (10 mmol/L KU-55933, Selleck- baseline p-Chk1 in the three cell lines with high A3A-A3B activity, chem), PARP inhibitor (AZD2281, Selleckchem), and Wee1i but not that in the three lines with low A3A-A3B activity, was (MK-1775, Selleckchem). When various inhibitors were used in reduced by knockdown of A3A and A3B (Fig. 1B and C). In combination with ATRi, they were added to cell cultures at the contrast to Chk1, Chk2, a substrate of ATM, was not phosphor- same time as ATRi unless indicated otherwise. ylated in an A3A-A3B–dependent manner (Fig. 1B). Thus, unlike

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A B 1.75

siCTL siA3A-A3BsiA3A-A3B #1 #2 siCTL siA3A-A3BsiA3A-A3B #1 #2 siCTL siA3A/BsiA3A-A3B #1 #2 1.50 pChk1 pChk1 pChk1

pChk2pChk2 pChk2 1.25 A3B A3B A3B 1.00 Chk1 Chk1 Chk1 NCI-H-2347 TOV21G OVCAR5 0.75 C

0.50 L L CT siCT siA3A-A3BsiA3A-A3B #1 #2 si siA3A-A3BsiA3A-A3B #1 #2 siCTL siA3A-A3BsiA3A-A3B #1 #2 C to U Activity relative TOV21G 0.25 pChk1 pChk1 pChk1 * A3B * A3B * A3B 0.00 G Chk1 Chk1 Chk1 BSH C78 BICR6 OV21 BICR31 HSC-4 SKOV3 HC SKBR3 OV17R T OVCAR5 HCC78 SKBR3 OV17R NCI-H-2347

Figure 1. APOBEC3A and APOBEC3B activate ATR in cancer cells. A, Relative A3A-A3B activity in a panel of cancer cell lines. A3A-A3B activity was determined from 20 mgcell extracts as shown in Supplementary Fig. S2. The activity of TOV12G is defined as 1. Cell lines are ranked according to their overall A3A-A3B activity. Error bar, SD (n ¼ 3). B and C, The three high-A3A-A3B activity lines (B) and the three low-A3A-A3B activity lines (C) were transfected with control siRNA or A3A-A3B siRNAs. Levels of baseline p-Chk1 and p-Chk2 were analyzed by Western blot analysis. , a nonspecific band. in cells arrested by high levels of A3A (32), only ATR but not ATM are incompatible. Interestingly, ATRi did not affect the overall is modestly elicited by the endogenous A3A-A3B activity in A3A-A3B deaminase activity in cells (Supplementary Fig. S3E), proliferating cancer cells. These results imply that at levels toler- suggesting that A3A is not activated but acts differently upon ATR able in proliferating cancer cells, A3A and A3B primarily induce inhibition. In contrast to ATRi, inhibitors of ATM and DNA-PK replication stress but not DSBs. (ATMi and DNA-PKi), two other PI3K-like kinases involved in the DNA damage response (DDR), did not kill A3AWT-expressing cells ATR protects cells from APOBEC-induced replication stress preferentially (Fig. 2B). These results provide further evidence that To investigate the mechanism by which A3A and A3B induce A3A induces replication stress and that A3A activity creates a replication stress, we sought to conditionally express A3A or A3B specific reliance on ATR. in proliferating cells. Because A3A shares similar activity with A3B To understand how ATR protects cells against A3A-induced and is more potent than A3B in inducing genomic instability (17), replication stress, we analyzed the effects of ATR loss in A3A- we chose to express A3A in U2OS, a cell line widely used in the expressing cells. Treatment of A3AWT-expressing cells with a low studies of replication stress responses. As a negative control for concentration of ATRi that only partially inhibits ATR, or with wild-type (WT) A3A (A3AWT), the catalytically inactive A3A ATR siRNA, drastically increased gH2AX levels (Fig. 2C; Sup- mutant (A3AE72A) was also conditionally expressed (17). At the plementary Fig. S4A–S4C; ref. 35). The induction of gH2AX by levels expressed in these cell lines, neither A3AWT nor A3AE72A ATRi was not observed in A3AE72A-expressing cells (Fig. 2C). The significantly affected cell proliferation (Supplementary Fig. S3A). ATRi-treated A3AWT-expressing cells displaying intense gH2AX gH2AX, a DNA damage marker, was not detectable in these cell signals were also TUNEL (terminal deoxynucleotidyl transfer- lines (Supplementary Fig. S3B). However, a low level of p-Chk1 ase–mediated dUTP nick end labeling) positive, showing that was detected in cells expressing A3AWT but not A3AE72A (Fig. 2A). they were undergoing severe chromosome fragmentation (Sup- In contrast, Chk2 was not phosphorylated in A3AWT-expressing plementary Fig. S4D). In contrast to ATRi, neither ATMi nor cells (Fig. 2A). These results suggest that conditional expression of DNA-PKi induced gH2AX in A3AWT-expressing cells (Fig. 2D). low levels of A3AWT in cycling cells induces replication stress but To determine when A3AWT and ATRi induce DSBs during the cell not DSBs, recapitulating the impact of endogenous A3A-A3B in cycle, we sorted cells according to DNA content, chromatin- cancer cells. bound PCNA, and gH2AX staining (Fig. 2E). The cells displaying ATR plays a critical role in protecting cells against DNA repli- gH2AX signals were also positive for PCNA, suggesting that cation stress. Indeed, the ATRi VE-821 (33) killed cells more DSBs arise in the S-phase. Together, these results suggest that the effectively when A3AWT was induced (Fig. 2B). When A3AWT was action of A3A modestly elicits ATR, which in turn suppresses expressed in an ovarian cancer cell line (SK-OV3) harboring low A3A-induced DSBs during DNA replication via a feedback loop. endogenous A3A-A3B activity, it also increased the sensitivity to VE-821 (Supplementary Fig. S3C). A second ATRi, AZ20 (34), also APOBEC3A-induced replication stress is unique killed A3AWT-expressing cells preferentially (Supplementary Fig. In addition to ATRi, inhibitors of several other checkpoint or S3D). VE-821 preferentially killed cells expressing A3AWT but not DDR proteins, such as PARP, Chk1 and Wee1, are known to A3AE72A (Fig. 2B), suggesting that A3A activity and ATR inhibition preferentially kill cancer cells under replication stress (36).

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B A A3AWT WT E72A 100 A3AWT + DOX A3AE72A A3A A3A A3AE72A+ DOX – ++– DOX pChk1-pS317

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Figure 2. ATR counters APOBEC3A-induced replication stress. A, U2OS-derived inducible cell lines of A3AWT and A3AE72A were induced with doxycycline (DOX) or left uninduced. Levels of p-Chk1 and p-Chk2 were analyzed by Western blot analysis. B, Cells expressing A3AWT or A3AE72A were treated with ATRi, ATMi, or DNA-PKi as indicated. Cell survival was analyzed after 5 days of continuous treatments. Error bar, SD (n ¼ 3). C and D, Levels of gH2AX in cells expressing A3AWT or A3AE72A after 8 hours of treatments with ATRi, ATMi, or DNA-PKi. E, Cells were induced to express A3AWT or left uninduced and treated with DMSO or ATRi (3 mmol/L) for the indicated amounts of time. Levels of chromatin-bound PCNA, gH2AX, and DNA contents of 4,000 cells were quantiﬁed and plotted along the three indicated axes. Cells were colored according to the intensity of gH2AX staining.

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Furthermore, several replication inhibitors and DNA-damaging Given that Chk1 is a downstream effector of ATR in the drugs, such as hydroxyurea (HU), aphidicolin (APH), mitomycin replication stress response, it is surprising that ATRi is more C (MMC), methyl methanesulfonate (MMS), and camptothecin selective than Chk1i toward A3AWT-expressing cells. Even when (CPT), also kill cells by interfering with replication. To further Chk1i was used at low concentrations, continuous Chk1i treat- understand the nature of A3A-induced replication stress, we ments were unable to distinguish cells with or without A3AWT performed a "miniscreen" using cells with or without A3AWT (Supplementary Fig. S4E). However, compared with uninduced against a panel of replication inhibitors and DNA-damaging cells, cells induced to express A3AWT were more sensitive to drugs. Surprisingly, none of HU, APH, MMC, MMS, and CPT transient Chk1i treatments (Supplementary Fig. S4F). These preferentially killed A3AWT-expressing cells as ATRi did (Fig. 3). results suggest that Chk1i also has some selectivity toward Moreover, none of the inhibitors of PARP, Chk1, and Wee1 A3AWT-expressing cells, although it is not as strong as that of showed a clear selectivity toward A3AWT-expressing cells as ATRi ATRi. We recently showed that ATRi is more selective than Chk1i did (Fig. 3). toward cells under high replication stress due to activation of the

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0 0 0 543210 0.0 0.5 1.0 0.0 0.5 1.0 ATRi (μmol/L) Chk1i (μmol/L) Wee1i (μmol/L)

Figure 3. APOBEC3A-expressing cells are speciﬁcally sensitive to ATR inhibitor. U2OS-derived inducible cell lines of A3AWT and A3AE72A were induced with doxycycline (DOX) or left uninduced and treated with increasing concentrations of the indicated inhibitors or DNA-damaging agents. Cell survival was analyzed after 5 days of continuous treatments. Error bar, SD (n ¼ 3).

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A DMSO 8 h ATRi + DOX

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siUNG2#1 + DOX Relative cell survival (%) siUNG2#2 siUNG2#2 + DOX siUNG2#3 siUNG2#3 + DOX 0 0 0 543210 ––+– ––ATRi TL L CT 8 ATRi (μmol/L) siC si TL siC siCTL siCTL siREV3siAPE1 siUNG2#1siUNG2#2siUNG2#3 siUNG2#1siUNG2#2siUNG2#3 siRAD1 + E ATRi ATRi DOX DOX DMSO 8 h ATRi + DOX siCTL siCTL siRAD18 siREV3 siAPE1 0.1%10.9% 20.1% 27.4% 21.8% γH2AX PCNA Intensity (A.U.)

X A.U.) DNA Content (A.U.) γH2A

Intensity (

Figure 4. APOBEC3A induces DSBs through AP sites upon ATR inhibition. A, Inducible A3AWT cells were transfected with control siRNA or three independent UNG2 siRNAs and treated with DMSO or ATRi (3 mmol/L) þ doxycycline (DOX). Levels of chromatin-bound PCNA, gH2AX, and DNA contents of 4,000 cells were quantiﬁed and plotted along the three indicated axes. Cells were colored according to the intensity of gH2AX staining. B and C, Inducible A3AWT cells were transfected with UNG2 siRNAs and treated with increasing concentrations of ATRi (B) or with 5 mmol/L of ATRi (C) in the presence or absence of doxycycline. Cell survival was analyzed after 5 days of continuous treatments. Error bar, SD (n ¼ 3). D, Quantiﬁcation of gH2AX intensity in 2,000 inducible A3AWT cells treated with various siRNAs, ATRi, and doxycycline as indicated. E, Inducible A3AWT cells were treated and analyzed as in A except that RAD18, REV3, and APE1 siRNAs were used.

DNA-PK–Chk1 pathway upon ATR inhibition (35). It is possible The inability of these drugs/inhibitors to distinguish cells with or that the basal replication stress in U2OS cells is too high to reveal without A3AWT suggests that the replication stress induced by A3A the selectivity of Chk1i toward A3AWT-expressing cells. is unique and that ATR has a speciﬁc role in countering this stress The selective sensitivity of A3AWT-expresssing cells to ATRi is (see Discussion). surprising because cells defective in DNA replication, DNA repair, or the checkpoint are generally sensitive to multiple drugs/inhi- APOBEC3A induces replication stress through AP sites bitors in the panel above. If A3AWT induces replication stress in the To understand why A3A-induced replication stress is unique same way as some of the drugs/inhibitors, the effects of A3AWT and how ATR counters it, we next interrogated the induction of and these drugs/inhibitors should be additive and A3AWT expres- A3A-dependent DSBs by ATRi. APOBEC proteins catalyze C-to-U sion should render cells more sensitive to these drugs/inhibitors. changes in ssDNA, which are subsequently converted to AP sites

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by the uracil-DNA glycosylase UNG2 (31, 37). In S-phase cells gH2AX in ATRi-treated A3AWT-expressing cells (Fig. 6A and B), expressing A3AWT, the induction of gH2AX by ATRi was signifi- suggesting that A3A-induced replication stress arises from AP cantly reduced by knockdown of UNG2 (Fig. 4A; Supplementary sites. To test whether A3A induces AP sites at replication forks, Fig. S5A). UNG2 knockdown also partially suppressed the killing we used a biotin-labeled aldehyde-reactive probe (ARP) to of A3AWT-expressing cells by ATRi (Fig. 4B and C). These results visualize AP sites in cells (Fig. 6C; ref. 43). Neither A3AWT nor suggest that UNG2-generated AP sites are necessary for the induc- ATRi alone induced detectable ARP signals (Fig. 6D). However, tion of DSBs by ATRi. Notably, UNG2 knockdown also reduced significant levels of ARP foci were detected when both A3AWT the baseline p-Chk1 in A3AWT-expressing cells (Supplementary and ATRi were present. The ARP foci were dependent on A3A Fig. S5B), suggesting that AP sites are required for the activation of activity and UNG2 (Fig. 6D and E), confirming the specificity of ATR by A3AWT during replication. Thus, A3A-induced and UNG2- this assay for AP sites. Foci of AP sites were only detected in S- generated AP sites trigger modest ATR activation and promote phase cells, and they colocalized with PCNA (Fig. 6F; Supple- DSB formation when ATR is inhibited. mentary Fig. S6A and S6B), showing that AP sites accumulate at Several mechanisms are involved in the response to AP sites replication forks. Consistently, AP foci were reduced by CDC7 in the genome. During base excision repair (BER), AP sites are knockdown and roscovitine (Supplementary Fig. S6C and processed by the AP endonuclease APE1 and subsequently S6D), indicating the association of AP site formation with removed (38). In the S phase, translesion DNA synthesis (TLS) ongoing replication. APOBECs are known to act on ssDNA is important for DNA replication through abasic template, (19). Indeed, AP sites colocalized with RPA and the ssDNA allowing cells to tolerate AP sites (39, 40). To determine how detected by native BrdUrd staining (Fig. 6G). These results ATR suppresses AP site–induced DSBs, we tested whether suggest that ATR suppresses A3A-induced DSBs by limiting the ATR regulates APE1 or TLS. In contrast to ATR inhibition, action of A3A on ssDNA at replication forks (Fig. 6H). knockdown of APE1 or key TLS factors RAD18 and REV3 only Collectively, the results above suggest that ATRi promotes modestly increased gH2AX levels in A3AWT-expresssing cells formation of AP sites at replication forks by providing more (Fig. 4D; Supplementary Fig. S5C and S5D). In addition, ssDNA for A3A to act upon (Fig. 6H). The elevation of AP sites knockdown of APE1 or the TLS factors further increased gH2AX at forks impedes DNA polymerases (44), propelling a further levels in ATRi-treated A3AWT-expressing cells (Fig. 4E). These buildup of ssDNA and AP sites at replication forks, which ulti- results suggest that ATR suppresses AP site–induced DSBs mately drives cells into replication catastrophe. In this model, independently of BER and TLS. both A3A and ATRi are necessary drivers of a unique feed-forward To further investigate how ATR suppresses A3A-induced DSBs loop leading to replication catastrophe, explaining the synthetic during DNA replication, we analyzed PCNA monoubiquitination lethality between A3A activity and ATR inhibition. (PCNA-Ub), which is triggered by stalling of DNA polymerases (41). Although A3AWT alone induced a low level of p-Chk1 (Fig. The overall activity of endogenous APOBEC3A and APOBEC3B 2A), it did not detectably induce PCNA-Ub (Fig. 5A), suggesting renders cancer cells susceptible to ATRi that polymerase stalling is modest in the presence of functional As ATRi promotes formation of ssDNA, AP sites, and DSBs WT ATR. However, ATRi treatment of A3AWT-expressing cells, but not in cells induced to express A3A , we asked whether ATRi cells expressing A3AE72A, induced PCNA-Ub in a time-dependent exerts similar effects in cancer cells harboring high endogenous manner (Fig. 5A and B). Consistently, A3AWT alone did not induce A3A-A3B activities. The three cancer cell lines with high overall chromatin-bound RPA, which reflects the ssDNA resulting from A3A-A3B activity and the three cell lines with low A3A-A3B activity polymerase stalling (Fig. 5C). The chromatin-bound RPA in had similar fractions of S-phase cells (Fig. 1A–C; Supplementary A3AWT-expressing cells was significantly increased by ATRi, sug- Fig. S7A). In the three cell lines with high A3A-A3B activity, ATRi gesting that A3AWT induces substantially more polymerase stal- efficiently induced AP sites and gH2AX, both of which were ling upon ATR inhibition. The cells with high levels of chromatin- suppressed by knockdown of A3A and A3B (Fig. 7A and B). In bound RPA gradually became strongly gH2AX positive (Fig. 5D), TOV12G, ATRi induced replication catastrophe in cells with high indicating replication catastrophe driven by excessive ssDNA levels of ssDNA in a manner dependent on A3A (Supplementary accumulation (42). Partial knockdown of RPA, which increases Fig. S7B). In contrast, ATRi did not induce gH2AX in the three cell ssDNA exposure (42), significantly enhanced the induction of lines with low A3A-A3B activity (Fig. 7C). Furthermore, ATRi gH2AX even in absence of ATRi (Supplementary Fig. S5E and induced strong TUNEL signals in the three cell lines harboring S5F). If ATRi induces replication catastrophe in A3AWT-expressing high A3A-A3B activity, which were also suppressed by knockdown cells, this process should be suppressed by loss of CDC7 and CDKs of A3A and A3B (Fig. 7D). In contrast, ATRi did not increase (35), which reduces replication initiation. Indeed, the induction TUNEL signals in the three lines with low A3A-A3B activity in of PCNA-Ub, ssDNA, and gH2AX by ATRi in A3AWT-expressing an A3A-A3B–dependent manner (Supplementary Fig. S7C). cells was significantly reduced by CDC7 knockdown (Fig. 5E; Together, these results suggest that the overall activity of endog- Supplementary Fig. S5G–S5I). Roscovitine, a CDK inhibitor, also enous A3A and A3B in cancer cells renders them susceptible to decreased ssDNA and gH2AX (Fig. 5F; Supplementary Fig. S5J). ATR inhibition. Thus, when cells undergo robust replication in the presence of A3A, ATR inhibition triggers a cascade of polymerase stalling, Discussion ssDNA accumulation, and replication catastrophe. APOBEC3A and APOBEC3B impose a unique type of replication stress ATR suppresses accumulation of AP sites at replication forks Although DNA replication stress is known to be a major Given that A3A activates ATR and ATR suppresses A3A- source of genomic instability, how replication stress arises in induced DSBs, we next asked whether the action of A3A is cancer cells and whether there are different types of replication regulated by ATR. Knockdown of UNG2 reduced PCNA-Ub and stress is still incompletely understood (3). Previous studies

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**** A B C 30

A3AWT A3AE72A 0 h h h2 h4 8 h6 ATRi (3 μmol/L) − −− ++ ++ + DOX + ++ ATRi (3 μmol/L/8 h) 20 −− DOX PCNA-Ub +++ + PCNA-Ub PCNA (long expo) PCNA (long expo) 10 RPA Intensity (A.U.) PCNA (short expo) PCNA (short expo) Chromatin fraction Chromatin fraction 0 ––++ DOX ++–– ATRi (3 μmol/L/8 h) D A3AWT + DOX A3AE72A + DOX

DMSO 2 h ATRi 4 h ATRi 6 h ATRi 8 h ATRi 8 h ATRi γH2AX Intensity (A.U.) RPA Intensity (A.U.)

E F C DMSO ATRi + DOX

Ri + ROS siCTL siCTL siCDC7#1 siCDC7#2 ATRi AT − + ++ DOX γH2AX

A3AWT (Flag)

GAPDH γH2AX Intensity (A.U.) RPA Intensity (A.U.)

Figure 5. ATR inhibition in APOBEC3A-expressing cells leads to replication catastrophe. A and B, Levels of PCNA and monoubiquitinated PCNA in the chromatin fractions of ATRi-treated A3AWT or A3AE72A-expressing cells were analyzed by Western blot analysis. C, Levels of chromatin-bound RPA were analyzed in 1,000 inducible A3AWT cells after induction with doxycycline (DOX) and treatment with ATRi as indicated. Significance was determined by t test. , P < 0.0001. D, Cells expressing A3AWT or A3AE72A were treated with DMSO or ATRi (3 mmol/L) as indicated. Levels of chromatin-bound RPA and gH2AX in 5,000 cells were quantified. Cells with medium or high levels of RPA and gH2AX are divided by dotted lines and colored differently. The reduction of RPA in red cells compared with orange cells is likely due to severe chromosome fragmentation. E, Inducible A3AWT cells were transfected with control or CDC7 siRNAs, induced with doxycycline, and treated with ATRi (3 mmol/L) for 8 hours. Levels of chromatin-bound RPA and gH2AX in 3,000 cells were quantified. F, Cells were induced to express A3AWT or left uninduced, and treated with ATRi (3 mmol/L) and roscovitine (12 mmol/L) as indicated for 8 hours. Levels of gH2AX were analyzed by Western blot analysis.

have shown that alterations of origin firing and/or replication imposes a unique type of replication stress distinct from that fork progression associate with replication stress in cancer cells arising from other sources. (45–48). In addition, defects in the protection and/or restart of How A3A and A3B contribute to the replication stress in cancer stalled replication forks, such as those caused by loss of BRCA1, cells still requires further investigation. Although A3B is a nuclear BRCA2, and Fanconi anemia proteins, could also give rise to protein, A3A is primarily induced in cytoplasm in response to replication stress (7–11). Recent studies by others and us have foreign DNA or IFN (37, 49). A recent study suggested that implicated A3A and/or A3B activities in the mutagenesis in APOBEC3H haplotype I, rather than A3A, was responsible for cancer cells during DNA replication (12, 14, 15). However, the APOBEC mutations in the absence of A3B (50). Nonetheless, whether this process of mutagenesis imposes replication stress the mutation signature of A3A is prevalent in several cancer types is not known. In this study, we show that A3A indeed induces (24). The A3A-B fusion gene encoding A3A is associated with replication stress in a catalytic activity–dependent manner. increased cancer risk and the APOBEC mutation signature in Importantly, the A3A-induced replication stress renders cells tumors (25–29). Furthermore, A3A is upregulated in a subset specifically sensitive to ATRi, but not to a variety of replication of leukemia (30). All these findings suggest that A3A indeed inhibitors and DNA-damaging agents, suggesting that A3A affects the genome in cancer cells. Our studies suggest that the

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A B

DOX ATRi + DOX

siCTL siCTL siCTL siUNG2#1 siUNG2#2 siUNG2#3 siUNG2#1siUNG2#2siUNG2#3 − +++ + DOX + ATRi PCNA-Ub PCNA (long expo) PCNA

(short expo) γ H2AX Intensity (A.U.) Chromatin fraction RPA Intensity (A.U.)

C F DAPI AP sites PCNA Merge

base base base P O P O Biotin P O NH2 Cy3 O Streptavidin ARP P O OH P OH H P OH H Biotin O O N base base base P O P O P O

DEG E72A DAPI AP sites ssDNA(BrdUrd) Merge WT A 14 A3A A3 20

12 15 10

8 10

6 population (%) DAPI AP sites RPA Merge +

4 5 PCNA 2 Fraction of AP site–positive cells (%) Fraction of AP site–positive cells in 0 0 − + − + + ATRi − + + + + DOX/ATRi L L −− DOX CT CT ++ + si si siUNG2#1siUNG2#2siUNG2#3 H

APOBEC3A UNG2

DSBs & Replication ssDNA AP Sites BER (APE1) catastrophe

TLS Replication (RAD18, REV3) stress ATRi

Figure 6. ATR inhibition increases AP site accumulation at replication forks. A and B, Cells were induced to express A3AWT or left uninduced, transfected with control siRNA, or three independent UNG2 siRNAs, and treated with DMSO or ATRi (3 mmol/L) for 8 hours. DOX, doxycycline. Levels of PCNA and monoubiquitinated PCNA in the chromatin fractions were analyzed by Western blot analysis in A, and levels of chromatin-bound RPA and gH2AX in 5,000 cells were quantiﬁed in B. C, A schematic diagram of the chemical reaction in which an ARP binds to an AP site in DNA. ARP probes were detected with Cy3-conjugated streptavidin. D and E, Cells were transfected with control or UNG2 siRNAs (E), induced to express A3AWT,A3AE72A, or left uninduced, and treated with ATRi (3 mmol/L) for 8 hours. Fractions of AP site–positive cells in total cell populations (D) or PCNA-positive cell populations (E) were quantiﬁed. Error bar, SD (n ¼ 3). F, The AP sites induced by A3AWT and ATRi (3 mmol/L for 8 hours) colocalize with PCNA, a marker of replication forks. G, The AP sites induced by A3AWT and ATRi (3 mmol/L for 8 hours) colocalize with ssDNA (native BrdUrd staining) and RPA. H, A model that explains how A3A and ATRi drive a feed-forward loop that generates AP sites, elevates DNA replication stress, and promotes replication catastrophe. overall activity of endogenous A3A and A3B in cancer cells renders show that in cancer cell lines with high overall A3A-A3B activity, them susceptible to ATRi. A3B was recently shown to be tran- this activity is responsible for the baseline p-Chk1, suggesting that scriptionally induced by certain oncogenic events and DNA- A3A-A3B activity is the cause of replication stress. It is possible that damaging drugs that interfere with replication (51). Our results A3A and/or A3B are both inducers and effectors of replication

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ACNCI-H-2347 TOV21G OVCAR5 B L 5 15 6 CT siCTL siCTL siA3A-A3BsiA3A-A3B #1 #2 si siCTL siA3A-A3BsiA3A-A3B #1 #2 ++ + ATRi ++ + ATRi 4 γH2AX γH2AX 10 4 3 A3B * A3B * 2 population (%) population (%) GAPDH GAPDH population (%)

+ + 2 5 + NCI-H-2347 1 HCC78 PCNA PCNA PCNA 0 0 0 Fraction of AP site–positive cells in Fraction of AP site–positive cells in Fraction ++ ATRi ++ ATRi of AP site–positive cells in Fraction ++ ATRi L L L L CT CT CT L L L L siCTL siCTL siCT si si si siCT siCT siA3A-A3BsiA3A-A3B #1 #2 siCT siCT siA3A-A3BsiA3A-A3B #1 #2 ++ + ATRi ++ + ATRi siA3A-A3B #1 siA3A-A3B #1 siA3A-A3B #1 γH2AX γH2AX NCI-H-2347 TOV21G OVCAR5 A3B A3B D * 0.4% 4.1% 2.1% 20 20 0.7% 9.1% 4.5% 20 0.2% 7.8% 0.7% GAPDH GAPDH TOV21G SKBR3

10 10 10 L CT si siCTL siA3A-A3BsiA3A-A3B #1 #2 siCTL siCTL siA3A-A3BsiA3A-A3B #1 #2 ++ + ATRi ++ + ATRi TUNEL Intensity (A.U.) TUNEL Intensity (A.U.) TUNEL Intensity (A.U.) γH2AX γH2AX 0 0 0 ATRi ATRi * ++ ++ ++ ATRi A3B * A3B L L CT siCTL si siCT siCTL siCTL siCTL GAPDH GAPDH

siA3A-A3B #1 siA3A-A3B #1 siA3A-A3B #1 OVCAR5 OV17R

Figure 7. APOBEC3A and APOBEC3B render cancer cells susceptible to ATRi. A, The three cell lines with high overall A3A-A3B activity were transfected with control siRNA or A3A-A3B siRNA and treated with ATRi (6 mmol/L) for 8 hours. Levels of AP sites were analyzed using biotinylated ARP. Error bar, SD (n ¼ 3). B and C, The three cell lines with high overall A3A-A3B activity (B) and the three lines with low A3A-A3B activity (C) were transfected with control siRNA or A3A-A3B siRNAs and treated with ATRi (6 mmol/L) for 8 hours. Levels of gH2AX were analyzed by Western blot analysis. , a nonspeciﬁc band. D, The three cell lines with high overall A3A-A3B activity were transfected with control siRNA or A3A-A3B siRNA and treated with ATRi (6 mmol/L) for 8 hours. A.U., arbitrary unit. TUNEL signals of 2,000 cells were quantiﬁed for each condition.

stress in cancer cells, generating a feed-forward loop to promote unique in several ways. In APOBEC-expressing cells, ATR counters genomic instability and drive tumorigenesis. intrinsic mutators but not defects in DNA replication or repair Why and how is the A3A-A3B–induced replication stress machineries. Furthermore, ATR not only suppresses the induction unique? We and others recently showed that APOBEC-induced of AP sites by APOBECs, but also responds to the replication stress mutations are associated with the lagging strand of replication induced by AP sites. As ssDNA is a substrate of APOBECs, ATR forks, suggesting that these mutators preferentially target ssDNA apparently acts as a brake to the feed-forward loop driven by on the lagging strand (12, 14, 15). The replication stress induced APOBEC activity. When ATR is inhibited in APOBEC-expressing by A3A is dependent on UNG2, which generates AP sites follow- cells, the elevation of origin firing results in an increased number ing the cytosine deamination by A3A. The AP sites on the lagging of replication forks and increased amounts of ssDNA at the forks. strand interfere with DNA polymerases a and d (44), leading to Such a surge of ssDNA in the genome would allow APOBECs and increased ssDNA exposure and thus providing more substrate to UNG2 to generate more AP sites at replication forks, leading to A3A. Unlike other types of replication impediments, preferential additional replication stress, more ssDNA, and a further buildup stalling of polymerases on the lagging strand may result in of AP sites. This ssDNA and APOBEC-dependent feed-forward accumulation of ssDNA gaps without halting fork progression loop would ultimately drive cells into replication catastrophe (52). The self-perpetuating nature of APOBEC-induced replica- (Fig. 6H). tion stress, and/or its bias toward the lagging strand, may distin- It is worth noting that the effects of ATRi on APOBEC- guish it from other types of replication stress. expressing cells are distinct from those of replication inhibitors. Although HU and APH also induce ssDNA as ATRi does, they ATR counteracts an ssDNA and APOBEC-driven do not block the ATR-mediated replication stress response. In feed-forward loop the presence of functional ATR, HU or APH treatment of Our findings also suggest that ATR plays a crucial role in APOBEC-expressing cells would trigger the ATR-mediated feed- countering the A3A-A3B–induced replication stress. Although back loop to restrict ssDNA accumulation, which prevents cells ATR is generally important for the replication stress response, its from going into replication catastrophe. The unique abilities of specific role in suppressing APOBEC-induced replication stress is ATRi to induce ssDNA and block the ATR-mediated replication

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stress response may underlie its distinctive effects on APOBEC- that can be exploited therapeutically remains a critical question to expressing cells. Although replication inhibitors do not prefer- be addressed in clinical settings. It is conceivable that assays for the entially kill APOBEC-expressing cells, they may promote APO- A3A-A3B activity in cancer cells may broaden the clinical use of BEC-mediated mutagenesis in cancer cells by inducing ssDNA. ATR inhibitors and improve their efficacies in cancer therapy. It would be interesting to test whether an increase of APOBEC- mediated mutagenesis renders cancer cells more responsive to Disclosure of Potential Conflicts of Interest immunotherapy. No potential conflicts of interest were disclosed.

Targeting cancers harboring high APOBEC3A and APOBEC3B Authors' Contributions activities with ATRi Conception and design: R. Buisson, L. Zou Although expression of high levels of A3A leads to DSBs and Development of methodology: R. Buisson, L. Zou cell-cycle arrest, cancer cells expressing A3A and/or A3B are able to Acquisition of data (provided animals, acquired and managed patients, proliferate despite the accumulation of APOBEC mutations in provided facilities, etc.): R. Buisson, C.H. Benes, L. Zou their genomes. We show that the endogenous A3A and/or A3B in Analysis and interpretation of data (e.g., statistical analysis, biostatistics, cancer cells, even at levels insufficient to arrest the cell cycle, computational analysis): R. Buisson, M.S. Lawrence, L. Zou Writing, review, and/or revision of the manuscript: R. Buisson, C.H. Benes, induce modest levels of replication stress and confer ATRi sus- L. Zou ceptibility. Although A3A expression alone does not induce Study supervision:: L. Zou significant levels of ssDNA, it primes cells for the ssDNA-forming feed-forward loop that can be activated by ATR inhibition. The Acknowledgments endogenous A3A and/or A3B in cancer cells likely confer ATRi We thank members of the Zou and Dyson laboratories for helpful susceptibility in a similar manner. Although our results show that discussions. ATRi is more selective than Chk1i toward U2OS cells expressing A3A, we do not exclude the possibility that Chk1i can selectively Grant Support kill APOBEC-expressing cancer cells in other contexts. In cancer R. Buisson was supported by an NIH Pathway to Independence Award cells with low baseline levels of replication stress, A3A and/or A3B (1K99CA212154) and was supported by a Susan G. Komen Fellowship activity may confer susceptibility to both ATRi and Chk1i. (PDF16380839) and a Marsha Rivkin Scholar Award. This work was also It is important to note that overall A3A-A3B activity in cancer supported by grants from the Department of Defense (W81XWH-14-1-0082 cells is only one of the several factors affecting ATRi sensitivity. For to L. Zou), NIH (GM076388 and CA197779 to L. Zou), Breast Cancer Alliance example, it has been shown that loss of ATM, activation or (L. Zou), and the Wellcome Trust (102696 to C.H. Benes). fi RAS, MYC cyclin E The costs of publication of this article were defrayed in part by the payment of ampli cation of ,or , reliance on the alternative advertisement fi page charges. This article must therefore be hereby marked in telomere-lengthening (ALT) pathway, and speci c defects in DNA accordance with 18 U.S.C. Section 1734 solely to indicate this fact. repair can confer ATRi sensitivity (33, 35, 53–56). Whether the A3A-A3B activity in cancer cells, either alone or in combinations Received December 20, 2016; revised March 15, 2017; accepted May 2, 2017; with other oncogenic events, is sufficient to create a vulnerability published OnlineFirst July 11, 2017.

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OF12 Cancer Res; 77(17) September 1, 2017 Cancer Research

APOBEC3A and APOBEC3B Activities Render Cancer Cells Susceptible to ATR Inhibition

Rémi Buisson, Michael S. Lawrence, Cyril H. Benes, et al.

Cancer Res Published OnlineFirst July 11, 2017.

Updated version Access the most recent version of this article at: doi:10.1158/0008-5472.CAN-16-3389

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APOBEC3A and APOBEC3B Activities Render Cancer Cells Susceptible to ATR Inhibition Remi Buisson1, Michael S

APOBEC3A and APOBEC3B Activities Render Cancer Cells Susceptible to ATR Inhibition Remi Buisson1, Michael S